Cow's milk neutralizes the cytotoxicity of acrolein, a putative carcinogen in cigarette smoke

Abstract

Cigarette smoke is a strong and independent risk factor for esophageal cancer, while the consumption of cow's milk has been proposed as a protective factor. The mechanistic role of milk in preventing cancer, however, has not been clarified. We focused our study on acrolein, an abundant unsaturated aldehyde present in cigarette smoke. Acrolein is a highly toxic compound and a putative carcinogen. Using a cell culture system, we found that (1) acrolein caused necrosis in Ramos Burkitt's lymphoma cells, (2) the necrosis was inhibited by preincubation of acrolein with milk, and (3) acrolein formed adducts with milk proteins. These results indicated the protective effects of cow's milk against acrolein-induced cytotoxicity via protein-acrolein adduct formation.

Keywords: acrolein; cow’s milk; cytotoxicity.

Fig. 1.

Necrosis is induced by acrolein treatment. (A) Ramos cells were treated with acrolein (Acr, 100 µM for 4 hr) or staurosporine (Sta, 1 µM for 8 hr) and examined under microscope after mixing with Hoechst 33342 (1 µg/ml) and trypan blue (0.2%). Upper panels: phase contrast microscopic images, lower panels: fluorescent microscopic images. Ctlr: control Ramos cells. Scale bar: 100 µm. (B) DNA fragmentation was not detected in Ramos cells treated with acrolein (Acr, 100 µM) for 0, 2, and 4 hr (lanes 1–3, respectively) but was with the apoptosis-inducing reagent staurosporine (Sta, 1 µM) for 8 hr (lane 4). Cell viabilities determined by trypan blue dye exclusion test were 100, 70, and 40% (lanes 1–3, respectively). Lanes 5 and 6 are 100 bp- and 1 kb-DNA ladder makers, respectively. (C) Western blot analysis of PARP (top) and GAPDH (bottom). Samples correspond to those in Fig. 1B. Data are representative of two to three independent experiments with similar results.

Fig. 2.

Milk neutralizes acrolein toxicity. (A) Acrolein (Acr) was preincubated with various amounts of milk at 37°C for 15 min and the mixed samples were cultured with Ramos cells (0.5 × 10⁶ cells) at 37°C. The final concentration of acrolein was 100 µM. After 24 hr, viable cell number was determined by trypan blue dye exclusion test. The amounts of milk were 0, 0, 10, 20, 50 µl (lanes 1–5, respectively). The cell numbers in the acrolein-contained samples were shown in lanes 2–5 (+). (B) (C) Acrolein was preincubated with PBS or milk at 37°C for 15 min and the mixed samples were cultured with Ramos cells (0.5 × 10⁶ cells) at 37°C. The final concentration of acrolein was 100 µM. After 4 hr (B) or 24 hr (C), viable cell number was determined by trypan blue dye exclusion test. The amounts of milk were 0, 0, 20, 20 µl (lanes 1–4, respectively). The cell numbers in the acrolein-contained samples were shown in lanes 2 and 3 (+). Asterisks (*) indicate a statistically significant. *: P<0.01 (n=4) in (B), **: P<0.05 (n=4) in (C). n.s.: not significant. Tukey’s test was used for the analysis. (D) Western blot analysis of acrolein-adduct formation. Increasing amounts of acrolein were incubated with milk at 37°C for 30 min (lanes 2–4). The acrolein concentrations were 0, 100, 500 and 1,000 µM (lanes 1, 2, 3, and 4, respectively).